The invention relates to a novel prokaryotic expression system and proteins expressed thereby.
The industrial production of proteins has, in many instances, exploited the native expression and secretory systems of microorganisms and specifically bacteria. For example and without limitation the bacterium Bacillus subtilis (B.subtilis) is known to produce and secrete a number of proteins. One family of these proteins, xcex1-amylases, is of industrial importance and therefore the harvesting of this secreted protein is an activity currently undertaken by industry. However, the yield of some xcex1-amylases is significantly reduced by protein degradation during an/or following passage through the cell membrane.
It therefore follows that there is a need to provide a protein expression system which enhances the production of native and/or heterologous and/or recombinant protein and more specifically effectively enhances the secretion of protein from the cell.
The expression and secretion of heterologous and/or recombinant protein (i.e. proteins that are not native to that particular bacteria) typically involves transformation of a bacterial cell with heterologous DNA with a view to manufacturing or producing heterologous and/or recombinant proteins.
Microorganisms such as Escherichia coli (bacteria), Saccharomyces cerevisiae, Aspergillus nidulans and Neurospora crassa (fungi) have been used in this fashion. The expression of heterologous protein in primitive eukaryotes also allows some desirable eukaryotic post-translational modifications to occur in heterologous and/or recombinant proteins leading to an increase in the stability of the expressed proteins and subsequent improvement in yield. More recently the use of mammalian and insect cells have been developed to facilitate the expression of eukaryotic proteins that for various reasons cannot be expressed in a prokaryotic host cell.
However, the cost effectiveness of producing heterologous and/or recombinant protein still remains the major advantage offered by genetically engineered prokaryotic expression systems and indeed significant advances have been made in the development of genetically engineered E.coli strains that increase the yield of specific proteins. The development of these bacterial strains has also been married with an ever increasing development of more efficient vectors adapted to optimise the expression of recombinant protein. These vectors contain promoter elements that are genetically engineered to create hybrid promoters that can be switched on or off with ease.
However, there are three major disadvantages when using E.coli as a means of expressing heterologous and/or recombinant protein. Firstly, the high levels of expression lead to a precipitation of recombinant protein in the bacterial cytoplasm as xe2x80x9cinclusion bodiesxe2x80x9d. This feature was thought to be advantageous as it can provide a simple means of separating the insoluble recombinant protein from the soluble endogenous E.coli protein. However, in reality this advantage is not a general feature of the system as in many cases proteins remain an insoluble precipitate that can only be released into solution by using strong chaotropic agents. This presents a major problem if the protein in question is particularly labile and therefore loses biochemical or biological activity upon denaturation. Secondly, the expression of foreign protein in E.coli leads to rapid degradation of these proteins via an efficient proteolytic system. Thirdly, it is known by those skilled in the art that E.coli usually does not naturally secrete protein into its surrounding environment. Therefore, the purification of native, heterologous or recombinant protein has the major disadvantage that the desired protein has to be purified from endogenous E.coli protein.
E.coli strains have been engineered to allow the expression of recombinant proteins that would ordinarily be difficult to express in traditional laboratory strains of E.coli. However, these engineered E.coli strains are invariably not as biologically disabled as traditional laboratory strains of E.coli and as a consequence require containment levels that are higher than would normally be required.
The identification of alternative prokaryotic host cells and the development of means that facilitate the production of soluble, intact and biologically active protein is obviously desirable. However, notably the number of potential prokaryotic host cells is huge.
With a view to producing a novel protein and expression system we have chosen to genetically engineer, as our example, Bacillus, ideally B.subtilis, in order to provide an expression system that overcomes the problems of yield associated with prior art systems. We have focussed our attention on providing a Bacillus expression system that produces and ideally secretes protein(s) into the culture medium because this system enables an initial purification of the manufactured protein due to the absence of contaminating endogenous bacterial protein(s) and other macromolecules.
The biochemical composition of the B.subtilis cell wall is quite different from that of E.coli. The cell walls of E.coli and B.subtilis contain a framework that is composed of peptidoglycan, a complex of polysaccharide chains covalently cross-linked by peptide chains. This forms a semi-rigid structure that confers physical protection to the cell since the bacteria have a high internal osmotic pressure and can be exposed to variations in external osmolarity. In Gram-positive bacteria, such as the members of the genus Bacillus, the peptidoglycan framework may represent as little as 50% of the cell wall complex and these bacteria are characterised by having a cell wall that is rich in accessory polymers such as wall teichoic acids. In addition, teichoic acids may be attached to the outside of the cytoplasmic membrane in the form of lipoteichoic acids or membrane anchored wall teichoic acids.
Teichoic acids are simple polymers of alditol phosphate molecules linked to each other by phosphodiester bridges. The free hydroxyl groups of the alditol phosphate backbone may be occupied by alanine or sugar residues. The alanylation of teichoic acids has a major effect of neutralising the negative charge conferred by adjacent phosphate residues, thereby reducing the overall negative charge of the cell wall.
The cell wall therefore provides, amongst other things, protection to the cell membrane to prevent rupture. The peptidoglycan framework represents upto approximately 50% of the cell wall mass. The remaining wall material consists of components which differ significantly between Gram negative (E.coli) and Gram positive (B.subtilis) bacteria. B.subtilis, and many other Gram positive bacteria, is characterised by having a cell wall that is rich in the accessory molecule teichoic acid.
The alanylation of teichoic acids is controlled by the D-alanyl-lipoteichoic acid (dlt) operon, a cluster of five genes encoding proteins necessary for the alanylation of teichoic acid. The genes are termed dltA, dltB, dltC, dltD and dltE. With the exception of dltE, each of these genes have known functions, Perego et.al 1995, please see FIG. 1.
The partial or complete deletion of any individual member of the dlt operon, with the exception of the dltE, completely inhibits the alanylation of teichoic acid. However, there is no obvious phenotypic effect of deleting one or more of the dltA-D genes other than the inhibition of alanylation and consequential changes in the overall surface charge. Cell division and growth are apparently unaffected in B.subtilis. 
An unrelated gene, prsA, encodes a cell membrane located chaperone like molecule. The protein is involved in the folding of secreted proteins on the extracytoplasmic side of the cytoplasmic membrane (Kontinen et.al. 1991; Jacobs et.al 1993). Sequence homology with several peptidyl-prolyl-isomerases suggests that the PrsA protein is involved in the isomerisation of proline residues between cis and trans isomers in secreted proteins. A number of mutations have been identified and are relatively easy to determine by the diminished ability of prsA mutants to secrete xcex1-amylase. An example of one such mutation is prsA3, Kontinen and Sarvas, 1993. Interestingly although mutants possessing a mutation in prsA show a decrease in the secretion of xcex1-amylase and exoprotease, some secreted proteins, for example penicillinase, are unaffected. This suggests that PrsA is
selectively involved in the secretion of proteins and that this selection may be determined by the number/position of proline residues or nature of its nearest neighbours in secreted proteins.
In an attempt to identify second site suppressors of prsA3 we have randomly mutagenised B.subtilis with the mini-transposon, Tn-10. This transposon randomly integrates into bacterial DNA and, as long as an essential gene is not disrupted, the Tn10 mutants are viable.
We have taken a B.subtilis strain carrying the prsA3 mutation and identified Tn10 integration mutants that show enhanced secretion of xcex1-amylase into the culture medium. One such mutant, designated IH7231 was further analysed by DNA sequencing of the flanking regions of rescued Tn10 DNA to identify the site of integration. After sequence comparisons of the rescued DNA with the published B.subtilis genomic sequence we suprisingly found the rescued sequence to be homologous to the dltD gene of the dlt operon, Perego et.al. 1995.
The published prior art does not indicate an involvement of the dlt operon in the secretion of proteins from B.subtilis. Indeed the only apparent phenotypic change in B.subtilis cells disrupted for any of the dltA-D genes is the failure of the cell to add D-alanine to wall or lipo-teichoic acids. It is therefore both suprising and intriguing that the disruption of a dlt gene should have this phenotype.
It is therefore an object of this invention to develop a means of expressing recombinant protein in a prokaryotic expression system that allows the production of proteins and/or polypeptides in a biologically active form and at high concentration.
It is further object of the invention to develop a prokaryotic expression system that enables the secretion of native, heterologous or recombinant protein into culture medium to facilitate the purification of proteins and/or polypeptides that retains biological activity.
According to a first aspect of the invention there is provided a bacterial strain whose genome includes the dlt operon wherein the operon has been altered by substitution and/or deletion and/or insertion and/or mutation so that either production of at least part of at least one product(s)encoded by said dlt operon is prevented or at least part of at least one gene product is non-functional to the extent that the use of the strain to produce native, heterologous or recombinant protein is facilitated.
Reference hereto the term bacterial strain includes reference to any bacterial strain but ideally a Gram-positive bacterial strain and, more ideally, but not obligatory, a bacterial strain of the genus Bacillus.
It will be apparent to those skilled in the art that where heterologous and/or recombinant protein is to be produced the said bacterial strain will be transformed so as to include DNA encoding at least one selected heterologous and/or recombinant protein.
It will also be apparent to one skilled in the art that said alteration may be to at least one of the dlt A-E genes as represented in FIG. 1. So that said alteration ideally leads to a failure of said strain to add D-alanine to teichoic acid.
In a further preferred embodiment of the invention said alteration is to at least part of the dlt A-E genes.
In yet a further preferred embodiment of the invention said alteration is to at least part of dltA and/or dltB and/or dltC and/or dltD and/or dltE, preferaby dltB but ideally dltD.
It will be apparent that means to effect said alteration to the dlt operon are well known in the art. For example, and not by way of limitation, the insertion of genetic material into the dlt operon may be undertaken by transposon integration. Additionally or alternatively, the operon may be altered to provide for deletion of at least part of at least one gene located in the dlt operon by homologous recombination with at least one suitably designed vector and/or the replacing of at least part of at least one gene located in the dlt operon with homologous DNA carrying, for example, a translation termination codon thus preventing synthesis of a functional protein. Additionally or alternatively, the operon may be altered by base substitution and/or mutation by random or site-directed mutagenesis.
In yet a further preferred embodiment of the invention said dlt operon is altered by way of alteration of an expression control sequence, ideally a promoter, such that the promoter is made responsive to a specific signal, for example, the operon may be placed under the control of an inducible promoter such that expression of the operon encoded gene products may be selectively controlled.
It is well known in the art that means to place the aforedescribed genes under the control of regulatable promoters exist and include those means described for placing the dlt operon under regulatable expression.
The dlt operon is controlled by a single promoter element regulated by the transcription factor (sigma D or "sgr"D), therefore the above embodiment of the invention may comprise replacement of sigma D or "sgr"D with, for example, and not by way. of limitation, an IPTG inducible promoter. By placing the dlt operon under the control of an IPTG inducible promoter the expression of proteins encoded by the dlt operon can simply be induced by addition of IPTG.
Alternatively the expression of the dlt operon may be repressed by, for example and not by way of limitation, incorporation of a tetracycline responsive element. The tetracycline responsive element binds the TET repressor protein to prevent transcription from a promoter containing the responsive element. Therefore a bacterial strain according to the invention could be further genetically engineered to contain a gene expressing the TET repressor and a dlt operon containing the TET responsive element.
Methods to manipulate bacterial promoters in the aforedescribed manner are well known in the art.
In an alternative embodiment of the invention said alteration according to the invention involves manipulation of the native promoter element in a manner that results in the provision of a non-functional promoter element incapable of initiating transcription at the dlt operon.
In an alternative embodiment of the invention said alteration of an expression control sequence is an alteration to at least one mRNA stabilising sequence element located in non-coding regions of the dlt operon. More ideally still, said non-coding regions are located in the 5xe2x80x2 or 3xe2x80x2 non-translated regions of mRNA molecules encoded by the dlt operon.
It is well known in the art that the stability of bacterial mRNA is controlled to a greater extent by sequences located at the 3xe2x80x2 end of mRNA which interact with proteins to either stabilise or de-stabilise mRNA molecules. The selective deletion, substitution, insertion or mutation of the sequences may de-stabilise MRNA molecules derived from the dlt operon but in any event results in decreasing and/or inhibition in the expression of dlt encoded proteins.
In yet a further preferred embodiment of the invention said alteration of an expression control sequence is to 5xe2x80x2 translation control sequences of mRNAs encoded by the dlt operon.
Translation control sequences are well known in the art and include, by example and not by way of limitation, Shine Dalgarno sequence motifs found near the translation start codon in many prokaryotic mRNA""s.
In a third aspect of the invention there is provided a method for producing a desired native, heterlogous or recombinant protein and/or polypeptide, wherein bacteria, as aforedescribed, is used for the production of the protein and/or polypeptide by;
i optionally, transforming a bacterial strain according to the invention with a suitable vector genetically engineered to facilitate expression of said polypeptide;
ii culturing said bacterial strain under conditions conducive to the production of said polypeptide; and
iii recovering and purifying the said polypeptide from said bacterial strain and/or growth medium.
The introduction of a vector into a bacterial strain according to the invention may be by any method known in the art, such as conventional transformation methods, electroporation, conjugation or protoplast transformation. The expression construct may be a plasmid or any other vector suitable for the specific method used for introducing said expression construct into a bacterial cell.
In essence the invention provides a bacterial strain, ideally a B.subtilis strain, that has been mutated to provide a bacterial strain that is facilitated in the secretion of native, heterologous or recombinant protein into surrounding growth medium.
An embodiment of the invention will now be described by way of example only with reference to the following figures wherein: